EXPRESSION OF BRUCELLA GENES REQUIRED FOR LIPOPOLYSACCARIDE PRODUCTION UNDER THE CONTROL OF ARAC AND PTAC PROMOTERS

by Han, Shuo

Abstract (Summary)

Brucella spp. are gram-negative, facultative intracellular pathogen that causes an infectious and contagious bacterial disease, brucellosis, in humans and animals. Previous research showed that lipopolysaccaride (LPS) is a classically demonstrated virulence mechanism in Brucella. The main objective of this project was to design an inducible system that would allow or prevent the expression of LPS using the manBA genes which encode components of LPS through the regulation of different promoters, pTac and araC. This will allow us to better understand the significance of LPS expression and its link to virulence. If we are able to regulate the expression of LPS in smooth and rough strains, we will be able to further understand these differences and eventually help to create attenuated vaccines against brucellosis.
Construction of manBA expression vectors using pTac and araC promoters was carried out in the E. coli strain, DH10B as well as the conditions required for induction of manBA expression. The constructs were transferred into a Brucella melitensis (16M) manBA deletion mutant to determine optimal induction conditions. During the induction, specific time points were monitored for variations in manBA expression. Methods used to detect manBA expression include: acriflavine agglutination, SDS-PAGE followed by staining and Western Blots. Results showed that LPS expression could be regulated by pTac and araC promoters under specified induction conditions. Although the pTac promoter was found to be leaky, we were able to induce LPS expression in Brucella melitensis with the addition of glucose to the growth medium. The araC promoter construct was more tightly regulated than the pTac promoter and required DMEM, a defined media for expression. Therefore, we have developed two inducible systems which would aid in the study of LPS virulence mechanism in Brucella. Future work will include infecting murine macrophages using the inducible Brucella constructs to study intracellular trafficking and survival of the transformed bacteria.
Construction of manBA expression vectors using pTac and araC promoters was carried out in E. coli strain, DH10B The conditions required for induction of manBA expression were tested for the respective promoters in E. coli DH10B. The constructs were transferred into Brucella melitensis (16M) manBA-knockouts to carry out induction under experimentally determined conditions in Brucella. During the induction, specific time points were monitored for variations in manBA expression. Methods used to detect manBA expression include: Acriflavine agglutination, SDS-PAGE followed by staining and Western Blots. Results show that LPS expression can be regulated by pTac and araC promoters under specified induction conditions. Although the pTac promoter was leaky, we were able to induce LPS expression in Brucella melitensis with the help of glucose. The araC promoter construct was more tightly regulated than the pTac promoter and required DMEM, a defined media for expression. Therefore, we have developed two inducible systems which would aid in the study of LPS virulence mechanism in Brucella. We also used a special staining method of the SDS-PAGE to detect LPS production. Future work will include infecting murine macrophages using the inducible Brucella constructs to study intracellular trafficking and survival of the transformed bacteria.